Microstructure correlation counter

ABSTRACT

An apparatus for analyzing unknown microstructures in samples with known microstructure configurations, both biological and non-biological, by optically correlating the unknown with the known. The apparatus employs a microscope to produce images of unknown microstructures in a sample, a laser for illuminating the sample, a Vander Lugt correlator, including a preselected matched filter for particular known microstructures of interest, for optically correlating the image of the unknown microstructures with preexisting images of known microstructures, a CCD array to detect correlation indicia, and a microcomputer to control positioning of the sample for imaging, orienting the matched filter, and scanning by the CCD array of the indicia resulting from the correlation for reading and counting the same.

BACKGROUND OF THE INVENTION

The present invention is an optical correlation counter, and moreparticularly is a cell correlation counter. The specific inventiondescribed herein can be computer controlled.

There is a continuing need for counting of very small particles in avariety of environments. Specifically, in cell research there are manyareas which would benefit from more efficient counting techniques.Examples include cancer research, blood cell counting, sperm counting,AIDS research, fertility studies, and other areas of counting biologicalstructures, microstructures, and particles, as well as non-biologicalparticles and microstructures.

In older methods of cell counting performed in a laboratory environment,a human counter was required to be located at a microscope for extendedperiods of time to effectively, reliably and manually examine a sample.Even under the best of conditions in the laboratory environment, therequirement for carefully counting individual items of interest such assperm cells and blood cells was, and is, very tiring and laborious. Theprocess is time-consuming and subject to inconsistencies and variablessuch as operator experience and fatigue.

In addition to the fully manual cell or particle counting systemsrequiring varying degrees of human interaction, there have been avariety of systems in the prior art that have introduced some degree ofcomputer control to permit mechanization, or at least partialmechanization, of the counting activity. Thus, U.S. Pat. No. 702,595 toMutschler et al, describes a pattern recognition system with workingarea detection which automatically positions a field being examined forcell or particle counting in a proper position with respect to anoptical scanning means. This system is directed to optimization ofpattern recognition and to automatically enable examination of the fieldof the observation within a good working area. The system isparticularly applicable to examination of blood smears on a slide, bypositioning the slide to enable examination without operatorintervention.

Also, in U.S. Pat. No. 4,475,236 to Hoffman, a method is disclosed forrapidly analyzing a mixture of unknown stained cells and known cellshaving different staining characteristics, a cell at a time, in a flowcytometry system having a sample stream dimension in the range of theexpected cell dimensions. The cells are illuminated and fluorescence isdetected and related to the number of cells being observed. A histogramof the mixture sample may be analyzed by counting the cells in acontrolled population below a relatively low threshold value offluorescence intensity.

In U.S. Pat. No. 4,362,386 to Matsushita et al, a method is disclosedfor mechanization of a microscope stage to move the field of view of aslide having a blood smear thereon to an optimum position within thesmear, under computer control, so that white blood corpuscles can bedetected, counted, and automatically classified within the optimum areaof the slide.

Finally, in U.S. Pat. No. 4,213,036 to Kopp et al, a method of probing abiological cell sample with an optical source to determine thecharacteristics of the cell image by way of measuring parameters fromits two-dimensional Fourier transform is disclosed. This patentdiscloses a method of measuring discriminating parameters for cellclassification by use of the Fourier transform technique.

The aforementioned techniques of the prior art employ varying degrees ofmanual, semi-automated, and fully automated techniques for smallbiological particle examinations, classifications, and counting. None,however, employ the techniques and methods of the present invention.

SUMMARY OF THE INVENTION

It is thus an object of the present invention to provide a means andmethodology for counting recognized biological and non-biologicalmicrostructures.

It is yet another object of the present invention to provide a fast,reliable, and repeatable means and methodology for counting recognizedmicrostructures.

It is still another object of the present invention to provide asemi-automated means and methodology for counting recognizedmicrostructures.

In accordance with the present invention, a specimen sample containingmicrostructures, such as cells in human blood, is placed on the stage ofa microscope moveable in two-dimensions relative to the imaging assemblyof the microscope. The specimen is illuminated by a collimated lightsource such as a laser, and the image upon leaving the imaging portionof the microscope is communicated to an optical correlator, whichconsists of a Vander Lugt correlator which includes a matched-filterspatial light modulator for the particular type of specimen being soughtin the image being transmitted from the microscope. The matched-fiber islocated within the Fourier plane of the first lens of the correlator. Acharge coupled detector (CCD) array of a CCD camera is located in theimage plane behind a cross-correlation lens within the correlator toreceive correlation peaks, the indicia of correlation, resulting from amatch between the Fourier transform image of the image communicated fromthe specimen being observed by the microscope and the image constitutingmatched filter. The present invention, in its preferred embodiment,includes a microcomputer to position the specimen sample on thetwo-dimensionally moveable microscope stage for examination of variousfields of regard (FOR) preselected for the particular specimen beingexamined within the microscope field of view. The microcomputer alsocontrols the spatial light modulator, which is the matched filter withinthe optical path of the Vander Lugt correlator, in order to position thefilter in various spatial alignments relative to the specimen imagebeing transmitted by way of the microscope through the correlator, sothat all matching microstructure configurations and spatial orientationscan be accommodated by the invention. The invention does require thatmatched filters be prepared ahead of time and stored, if in electronicform, within the primary or peripheral memory of the microcomputer, oras positive or negative transparency images, so that they may beemployed as spatial light modulators for use within the correlator ofthe system. Finally, the microcomputer is used to programmably controlthe scanning of the correlator image plane by means of the CCD arrayemployed by the invention for detecting and counting of the indicia ofcorrelation, the correlation peaks appearing thereon. The apparatus andmethodology of the present invention thus make it possible not only toidentify and thus recognize a particular microstructure whether it bebiological or non-biological, but also to permit the counting of suchmicrostructures. The use of the optical Fourier transform techniques inthe present invention make possible high speed and accurate recognitionand counting considerably faster than the prior art technologies.

BRIEF DESCRIPTION OF THE DRAWINGS

The above recited objects and summary of the present invention will bemore readily understood by reference to the following detaileddescription when considered in conjunction with the accompanyingdrawings wherein:

FIG. 1 is a schematic block diagram of the present invention.

FIG. 2 is a schematic perspective representation of the correlator ofthe present invention.

FIG. 3 is an enlarged representation of a Fourier transformedmicrostructure image constituting the matched filter.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

With reference FIG. 1, the counter 10 of the present invention is shownin conjunction with the specimen sample 12 in the optical path of themicroscope 11. The counter 10 is comprised of the microscope 11 havingthe moveable stage 14 which is shown located upon the microscope base 15in line with the microscope imaging assembly 18. The sample 12 restsupon the reflector 13, a right angle prism, which is shown in positionupon the two-dimensionally-moveable X-Y stage 14 upon the microscopebase 15. The laser 16 illuminates the specimen sample 12 by providingcoherent light via the reflector 13. The light communicated to reflector13 is transmitted at an angle of 90 degrees upward through the specimensample 12 and into the microscope imaging assembly 18. The image of theilluminated sample 12 and the microstructures therein is communicatedfrom the microscope 11 by way of the corner reflector 20 to means 24 foranalyzing the unknown microstructures in the sample with knownmicrostructure configurations in accordance with the present invention.The correlator 24 consists of the Vander Lugt correlator 22 whichcontains the matched filter spatial light modulator 29 in the correlatoroptical path for the purpose of introducing a known and pre-storedmicrostructure Fourier transform image to the correlator 22 forcorrelation with the Fourier transform image of the coherentlyilluminated and microstructure containing specimen sample 12 receivedfrom the microscope 11. The CCD camera 33 is also located in theanalyzing means 24 in optical alignment with the cross correlation lens30, seen in FIG. 2, that is included within the Vander Lugt correlator22. The spatial light modulator 29 is connected electrically to themicrocomputer 34 so that the computer can provide electronically imagedand stored matched filters 32 to the analyzing means 24 in the varietyof orientations desired. Also, the CCD camera 33 within the analyzingmeans 24 is connected to the microcomputer 34 so that the results of thecross correlation function within the Vander Lugt correlator 22 can bedetected, counted, and passed to the computer for count storage. Thearrow going from the microcomputer 34 to the CCD camera 33 in theanalyzing means 24 represents the communication of control signals tothe camera to permit it to scan the entire cross correlation imagethrough as many fields of regard (FOR) desired for the purposes ofcounting the indicia of the correlated microstructures, the correlationpeaks within the original specimen image introduced to the correlator22.

More specific details of the correlator 22 are shown in FIG. 2 where aspherical Fourier transform lens 26 is shown coaxially aligned a focallength away from the spatial light modulator 29 also coaxially alignedin front of the cross correlation lens 30. The cross correlation lens 30is likewise located coaxially in front of the image plane 40 of thecorrelator 22 occupied by the charge-coupled device array 42 of the CCDcamera 33, or other scanning detector, and a focal length distance awayfrom that array.

OPERATION

In operation, the coherent illumination of the specimen sample 12 by thelaser 16 produces a normal image of the first fields of regard (FOR) 25,FIGS. 1 and 2, being imaged by the microscope imaging assembly 18. Themicrostructure images 27 within the FOR 25 are shown in FIG. 2 prior tothe Fourier transform lens 26. That FOR image 25 containing themicrostructure images 27 is produced at the Fourier transform lens 26which in turn produces fourier transformed images 28 at the spatiallight modulator 29 in FIG. 2. The image 28 and the matched filter 32 inFIG. 2 are in actuality in the same plane. They are shown apart fordiscussion and understanding only. An enlarged view of a singletransform microstructure image 28 which presents at the spatial lightmodulator 29 as the matched filter 32 is shown in FIG. 3.

Microcomputer 34 is programmed to position the moveable stage 14 ofmicroscope 11 upon microscope base 15 through a sequence oftwo-dimensional X-Y, horizontal plane adjustments in order to presentthe pre-determined number of FOR of the specimen sample 12 forexamination.

In the preferred embodiment of the present invention, the computer 34digitally stores the Fourier transform images 28 of the microstructureimages 27 of interest for use as match-filter spatial light modulators29. These pre-stored and transformed microstructure images 28 which arethe matched filters 32 are programmably rotatable by computer 34 througha number of planar orientations, relative to the FOR image 25,pre-determined to be necessary to attain the highest degree ofcorrelation needed. In the alternative, a rotationally invariant filtermay be employed for the matched filter spatial light modulator 29.

Thus for each field of regard (FOR) of the specimen sample 12 an imageis presented in sequence to the analyzing means 24. For each such FORimage 25 the matched filter 32 is programmably sequenced by computer 34through the pre-determined number of spatial orientations relative tothe FOR image 25 to maximize or optimize the opportunities forcorrelation in relation to the particular type of microstructure beingexamined, e.g., cancer cell, AIDS virus, etc. The correlation functionis performed repeatedly for each FOR of a specimen sample 12 until allFORs are exhausted. When a match of the Fourier transformedmicrostructure image 28 occurs at the match filter 32, the multipliedimage thus produced is then communicated to the cross correlation lens30, located a focal length in front of the image plane 40 of thecorrelator 22 where it is further Fourier transformed to an image at theimage plane 40 in FIG. 2. The image at the image plane 40 constitutesindicia of correlation or correlation peaks 31 resulting from matchingbetween the transformed microstructure images 28 and the matched filter32 at the spatial light modulator 29. These indicia or peaks arereadable and countable manually, semi-automatically or automatically.The CCD array 42 of the CCD camera 33, or equivalent image pixelscanning and capture devices and an electronic counter such as wouldtypically be found in the microcomputer 34 may be used forsemi-automatic and fully automatic counting.

It will be understood by those skilled in the art that the presentinvention may be configured in a variety of forms consistent with thedetails and operational description recited herein while remainingwithin the bounds of the claims which follow.

What I now claim as my invention is:
 1. An apparatus for countingmicrostructures in a sample by optical correlation of an image of saidmicrostructure with a pre-existing image of said microstructureproducing indicia of said correlation, said apparatus comprising:(a) amicroscope for imaging said sample having an optical axis; (b) aprogrammable stage being translatable in X-Y dimensions relative to saidoptical axis of said microscope and being capable of holding andpositioning in said X-Y dimensions all or portions of said sample in theoptical field of view of said microscope, said stage being operationallydisposed in juxtaposition with said microscope; (c) a laser aligned withsaid optical axis of said microscope and capable of coherentlyilluminating said sample and being operationally disposed injuxtaposition with said microscope to produce an image of themicrostructure in said sample; (d) a Vander Lugt correlator disposedcoaxially with said optical axis of said microscope, said correlatorhaving a spherical Fourier transform lens disposed to receive from saidmicroscope the sample image produced by said laser, a matched filterspatial light modulator disposed coaxially a focal length behind saidFourier lens for providing a pre-existing image of a particularmicrostructure, a cross-correlation lens disposed coaxially behind saidmatched filter spatial light modulator, and an image plane disposed afocal length behind said cross-correlation lens, said correlator beingoperable for optically correlating said image of the microstructure insaid sample with said pre-existing image and producing indicia of saidcorrelation which is received at said image plane thereof; (e) meansdisposed in optical juxtaposition with said correlator for detecting theindicia of correlation at said image plane; and (f) a computeroperationally disposed for programmable X-Y dimension translating ofsaid translatable stage holding said sample at said microscope, rotatingsaid matched filter spatial light modulator of said correlator,programmable scanning said detecting means across said image plane, andcounting the detected indicia of correlation received at said imageplane.
 2. The apparatus of claim 1 wherein said detecting means at saidimage plane contains a CCD array for detecting the indicia of saidcorrelation.
 3. The apparatus of claim 2, wherein said CCD array furthercomprises means for scanning said array pixel by pixel for counting theindicia of said correlation.
 4. The apparatus of claim 2 wherein saidimage plane is scanned by said CCD array.
 5. The apparatus of claim 1wherein said matched filter spatial light modulator is programmablyrotatable about the optical axis of said correlator with which saidfilter is coaxially disposed.